ENUCLEATION BY CENTRIFUGATION OF IN VITRO-MATURED BOVINE OOCYTES FOR USE IN NUCLEAR TRANSFER

Nuclear transfer has the potential to produce large numbers of identic al progeny, Current limitations of the technique are associated with t he use of micromanipulation for the demanding enucleation and reconsti tution procedure, With the overcoming of this limitation, increased nu mbers of nuclear transfer embryos could be produced. Centrifugation of bovine oocytes at 15 000 x g for 2 min resulted in the stratification of organelles within the cytoplasm, which positioned the metaphase II spindle for enucleation. After removal of the zona pellucida with Pro nase, the oocytes were centrifuged in a Percoll density gradient so th at the oocytes were stretched apart to form cytoplasts and the metapha se II spindle was separated from the majority of oocytes, Enucleation by centrifugation efficiently produced a consistent population of enuc leated cytoplasts from bovine in vitro-matured oocytes. The population of enucleated cytoplasts was enriched by exclusion of the cytoplasts that exhibited an extrusion cone containing metaphase II chromosomes 6 h after centrifugation, The enucleated oocyte cytoplasts were aggrega ted with blastomeres isolated from in vivo-collected morulae. The aggr egated embryonic cells were electrofused to obtain nuclear transfer em bryos that were placed into a sodium alginate false zona and were capa ble of cleavage and development in vitro. The development of nuclear t ransfer embryos produced through use of centrifugation and aggregation techniques was comparable with that of nuclear transfer embryos produ ced by micromanipulation techniques.